Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the invention pertains.
Factor VIII/vWf is a plasma coagulation complex composed of two differently sized, noncovalently linked, and genetically independent proteins. The higher molecular weight component that is involved in platelet-vessel wall interactions is termed von Willebrand factor (vWf). vWf is a multimeric glycoprotein which plays an essential role in the hemostatic process. It is deficient and/or defective in the inherited bleeding disorder von Willebrands disease (vWd). The lower molecular weight component, factor VIII-coagulant (VIII:C), is deficient and/or defective in the inherited disorder, hemophilia A. Many acquired disease states can also exhibit low levels of vWf and VIII:C. In contrast, elevated levels of vWf and VIII:C are associated with acute and chronic inflammatory diseases, cancers and thrombotic states.
The laboratory diagnosis of vWd is dependent on demonstrating decreased levels of plasma vWf. Measurement of plasma von Willebrand factor by electroimmunoassay (EIA) or radioimmunoassay (RIA) is currently an important clinical diagnostic and research tool to determine whether an individual is affected with von Willebrand's disease (vWd), one of the most common bleeding disorders of man and domestic animals. Recent estimates of the gene frequency of inherited, congenital vWd in humans range from 1 to 500 to as high as one in 50 live births of either sex, whereas in inbred purebred dog families, prevalence can be as high as 80% (Doberman pinschers) and varies between 15 to 45% in many other breeds. Furthermore, an acquired form of vWd is increasingly being recognized in association with autoimmune thyroiditis in humans and dogs.
In addition to the vWf deficiency states that lead to a clinically expressed bleeding tendency, vWf is known to be an important contributor to the development or promotion of cardiovascular disease. Monitoring vWf levels is thus valuable in both clinical and experimental assessment of thrombosis and atherogenesis, for example, in patients and for long-term cardiovascular and aging studies in captive primates.
Thus, as physicians and veterinary clinicians have become increasingly aware of the prevalence of vWd as a cause of or precipitating factor in bleeding, the demand for a practical and inexpensive screening test for vWf has arisen. Currently the EIA or RIA procedures utilized routinely require expensive equipment, potentially toxic reagents, sophisticated technology, and skilled technologists. These tests are labor-intensive assays which increase the actual and retail cost of the assay.
There is a need within the field for a sensitive assay which is quantitative, specific, easy to perform and has increased efficiency. Such an assay would be especially advantageous to the Veterinary Hematology Laboratory of the State of New York Department of Health, which routinely analyses vWf antigen levels by EIA of more than 10,000 samples per year of humans, dogs and other species.
In order to permit the measurement of trace amounts of platelet vWf for research purposes, and to improve the sensitivity of routine electroimmunoassay for plasma vWf, the subject invention provides a qualitative and quantitative, highly sensitive, specific, and reproducible enzyme-linked immunosorbent assay (ELISA) for vWf.
The ELISA of the subject invention utilizes antibodies from multiple species which can be used to detect von Willebrand factor antigen in multiple species.
Previous ELISAs that measure human vWf have been described in the literature. A significant disadvantage, however, of these procedures is the necessary rabbit and goat antibodies to vWf are raised against human vWf. Because the original source of material for preparing the immunogen is human plasma, it presents the danger of transmitting blood-borne diseases to other humans. The subject invention provides an assay which does not depend upon the utilization of vWf man plasma from human plasma to raise antibodies and, therefore, has little infectious potential.
In addition to humans and dogs, the assay of the subject invention can quantitatively determine vWf levels in many other domestic and laboratory animal species including but not limited to non-human primates, horse, pig, mouse, rat, guinea pig, cow and cat. Previously, accurate measurements of vWf were not possible for many of these species. Utilizing the ELISA assay of the subject invention applicants have diagnosed vWd for the first time in a valuable quarterhorse with a significant bleeding history. The subject assay can thus be used to screen valuable racing and working horse stock for the presence of vWd. For example, exercise-induced pulmonary hemorrhage in horses is a relatively common and serious problem of complex etiology that remains poorly understood.
The ELISA assay of the subject invention solves a long standing problem which has not been recognized by those working in the area of human von Willebrand factor. This long standing problem, however, has become apparent to applicants as researchers in the area of von Willebrand factor in connection with numerous species. The problem relates to the need for an assay which can be used to qualitatively and quantitatively detect von Willebrand factor antigen in multiple species without the need to create or purchase an assay which is specific for each individual species, for example, rat, guinea pig, mice, etc. It is impractical in the research area to have individual vWf assays for each species which may be encountered in the course of research relating to vWf. Thus, the diagnostic kits of the subject invention unexpectedly solve this long standing problem which has previously been unrecognized in the vWf field. This assay will be of particular use in research work where investigators can evaluate von Willebrand factor with a fast and efficient assay useful for each of these species. Furthermore, the ELISA assay of the subject invention is much more sensitive than previous EIA or RIA assays for vWf, and thus provides a definite advantage over the previously used conventional assays.
Research into developing assays for von Willebrand factor in the past which would quantitate vWf in such diverse multiple species have been unsuccessful, where the assay of the subject invention has succeeded.
Silveira et al. (36) have described a sandwich ELISA system to measure plasma von Willebrand factor antigen (vWf:Ag) in humans. The test utilizes antibodies to human vWf:Ag raised in rabbits. The rabbit antibody is immobilized in the wells of a microtiter plate and acts as the reactive capture surface for vWf:Ag. Serially diluted plasma samples containing either known or unknown levels of vWf:Ag are reacted with the immobilized antibody, and then quantified by detection with a second (sandwich) antibody to human vWf:Ag (goat antihuman vWf:Ag) and a horseradish peroxidase-conjugated porcine anti-goat IgG. The peroxidase activity generated by the final antibody is proportional to the captured amount of vWf:Ag and is measured by the color change it catalyses in the substrate orthophenylenediamine (OPD). The color change reaction is subsequently quenched with sulfuric acid and the optical density measured to determine the concentration of vWf:Ag in the plasma tested.
A disadvantage of the above process is that the rabbit and goat antibodies to vWf which function as the initial capture, immobilizing or anchor antibody and the second or sandwich antibody are raised against human vWf:Ag. Because the original source material for preparing human vWf:Ag is human blood, it presents the danger of transmitting blood-borne diseases to other humans. As a result, the test should only be employed in specially equipped and sanitized laboratories designated for handling specimens of human origin (37). Furthermore, this ELISA assay is not used to detect vWf:Ag in a wide variety of vertebrate species.
Zimmerman et al. (1) describe an immunoassay to quantify the plasma protein deficient in human vWd by utilizing a precipitating antibody prepared in rabbits. This development initiated a series of studies that led to a substantial increase in the understanding of the structure and function of the factor VIII - von Willebrand factor complex (2-4). Parallel studies conducted with animal models of hemophilia A and vWd led to similar findings (5-8). The measurement and identification of vWf:Ag in the plasma and cells of these animals required preparation of polyclonal antisera specific for the vWf:Ag of each species. The development of species-specific antibodies in rabbits and goats against dog (9,10), pig (7,11), rat (12), guinea pig (13), cow (14), and rabbit vWf:Ag (15,16) has been reported. As in Silveira et al., this assay is not used to detect vWf:Ag in a wide variety of vertebrate as is the assay of the subject invention.
Bennett and Ratnoff (17) reported precipitin reactions of partial identity between rabbit antihuman vWf:Ag and the plasmas of 15 different mammalian species, while Coppola et al. (18) demonstrated cross-reactivity of four nonhuman primate plasmas and 20 other mammalian plasmas in a two-site immunoradiometric assay using either human homologous or rabbit heterologous antibodies. Bouma et al. (19) published the first quantitative cross-species data whereby canine vWf:Ag was determined with rabbit antibodies specific for human vWf:Ag. It was subsequently found that species cross-reactivity permitted the measurement of feline (20) and equine vWf:Ag (21) with the anti-canine vWf:Ag reagent. Again, the diverse vertebrate species in which the subject assay can be used to measure vWf:Ag are not disclosed by these references, and thus do not provide the advantages, i.e. to researchers, which the subject assay provides.
Utilizing monoclonal antibodies, it has been demonstrated that cross-reactivity exists between antibodies to porcine (22) or bovine (23) vWf:Ag and human vWf:Ag, and between antihuman vWf:Ag and canine (24), porcine and bovine vWf:Ag (24,25).
Katzman et. al. (22) describe twenty monoclonal antibodies developed against porcine vWf antigen and indicate that six of these clones were also active against human vWf antigen. These antibodies were not used in a quantitative fashion, ELISA or otherwise, to measure human vWf antigen and information on other species cross-reactivity was not disclosed.
Bradley, Franco and Reisner (23) disclose two monoclonal antibodies to human vWf antigen which could be used to quantify vWf antigen in porcine, bovine and canine samples. No details, however, of the methodology for quantitation of the animal vWf was provided. Their assay used antibodies prepared against human material and therefore maintained the potential for infectious transmission. The safety feature of antibodies to non-human products, therefore, remains a benefit of the subject invention. An additional difference between a preferred embodiment of the subject invention and the assay of the Bradley et al. reference is that the technique in the reference is competitive and does not utilize a capture antibody.
As with polyclonal antibodies, none of these assays utilizing monoclonal antibodies disclose the diverse vertebrate species for which the assay of the subject invention can be used.
Peake and Bloom (38) disclose an immunoradiometric assay for von Willebrands factor which was not cross-species reactive.
Prior to the subject invention, most of the assays for von Willebrand factor discussed in the literature have been applied to humans successfully. However, when they are used to look at multiple species, they are not successful. This may be due to the fact that the assays are not of the proper configuration or the antibodies used do not recognize conserved epitopes.
Cross-species reactivity between humans and dogs is well known to experts in the field. For example, it has been published that anti-human vWf can be used to quantitate canine vWf by electroimmunoassay. However, qualitative cross-species reactivity between humans and at least 25 or 30 vertebrate species is not known and is not expected because classical assays to measure vWf, for example, the standard Laurell assay, have failed to allow the detection of vWf in these other multiple vertebrate species. This standard Laurell assay allows the measurement using anti-human vWf of dog, cat, monkey and primate vWf antigen. However, it does not allow the detection of horse, rat, guinea pig, pig, cow, rabbit, llama, camel, and manatee, for example.
Applicants have recognized that the problems in regard to recognizing vWf in multiple species are related to three things:
1. The antibody must be capable of reacting with conserved epitopes of the vWf antigen. PA1 2. The antibody must be present in the right configuration in the assay. In other words, the assay must allow the antibody to recognize these conserved epitopes. If the antibody is not present in the right configuration, even if it is capable of recognizing the conserved epitope, it will not do so. PA1 3. There is an advantage to using plasma instead of serum. The reactive epitopes of vWf are sometimes destroyed when serum is made. In humans, serum or plasma will work. However, in other species plasma is required to preserve reactive epitopes.
Applicants have provided a solution to these problems with the subject invention, which provides antibodies capable of recognizing the conserved epitopes, and an assay configuration which allows the antibodies to recognize the epitopes. The diverse number of vertebrate species for which the subject invention can be used to detect vWf:Ag provides a further definite advantage over previous methods for detecting vWf:Ag.